Innovare International Journal of Pharmacy and Pharmaceutical Sciences

Academic Sciences ISSN- 0975-1491 Vol 7, Issue 10, 2015

Original Article PHARMACOKINETIC DRUG INTERACTIONS OF GLICLAZIDE AND ITOPRIDE IN NORMAL AND DIABETIC RATS

RAMA RAO VUNNAM*1, SRIHARSHA S. N.2, V. V. RAJESHAM3 1Faculty of Pharmacy, Dr. K. N Modi University, Newai, Rajasthan, India, 2Faculty of Pharmacy, Mahathi College of Pharmacy, Madanapalle, Chittoor-Dist, AP, 3Vijaya College of Pharmacy, Munaganoor (V), Hayathnagar (M), Hyderabad Email: [email protected] Received: 30 Jun 2015 Revised and Accepted: 24 Aug 2015 ABSTRACT Objective: The present study was aimed to investigate the safety, reliability of Gliclazide and possible drug interaction with Itopride when they were administered as combination treatment. Methods: Studies were conducted in normal and streptozotocin induced diabetic rats with oral administration of selected doses of gliclazide, itopride and their combination. Blood samples were collected from rats by retro orbital/marginal ear vein puncture at regular intervals of time. All the blood samples were analyzed for pharmacokinetic parameters by HPLC method. Results: There was no significant difference in pharmacokinetic parameters of both Gliclazide alone and combination with itopride in healthy and diabetic rats on day 1 and day 8. Conclusion: Based on the results it can be concluded that the concurrent administration of these two drugs have potential benefit without any drug interactions in the effective management of diabetes and gastroparesis. Keywords: Drug interactions, Gliclazide, Itopride, Pharmacokinetics, Gastroparesis, Diabetes mellitus.

INTRODUCTION MATERIALS AND METHODS Diabetes mellitus is a metabolic disease in which there are Materials high blood sugar levels over a prolonged period. Diabetes mellitus is due to the pancreas not producing enough insulin or the cells of the Drugs and chemicals body not responding properly to the insulin produced [1]. Gliclazide and Itopride were procured from Matrix laboratories as a Normally, the muscles of the stomach, which are controlled by the gift sample. Streptozotocin (STZ) was purchased from Sigma Chemical vagus nerve, contract to break up food and move it through the Co. The glucose estimation (GOD-POD) kit (Excel diagnostic, gastrointestinal (GI) tract. Diabetes is the most common known Hyderabad) was procured from the drug store. All HPLC grade cause of gastroparesis. People with diabetes have high levels of solvents (methanol and water) were procured from Finar chemicals blood glucose, also called blood sugar. Over time, high blood glucose Ltd., Ahmadabad. All chemicals used were analytical grade. levels can damage the vagus nerve. Methods Gastroparesis is a syndrome characterized by delayed gastric Animal study emptying [2], in the absence of mechanical obstruction of the stomach. The cardinal symptoms include postprandial fullness Twenty four healthy Wistar rats were (Weighing 200-220 g) (early satiety), nausea, vomiting and bloating [3]. In one tertiary selected for this study, all the animals were healthy during the referral series, diabetes accounted for almost one third of cases of period of the experiment. All efforts were made to maintain the Gastroparesis [4]. Diabetes patients with gastroparesis develop animals under controlled environmental conditions (Temperature retinopathy, neuropathy, nephropathy, nutritional compromise, 25 °C, Relative Humidity 45% and 12 h alternate lights and dark impaired glucose control, and a poor quality of life [5]. cycle) with 100 % fresh air exchange in animal rooms, uninterrupted power and water supply. Rats were fed with standard Symptoms attributable to gastroparesis are reported by 5 to 12% of diet and water ad libitum. The protocol of animal study was patients with diabetes [6, 7, 8, and 9]. Gliclazide is an oral hypoglycemic approved by the institutional animal ethics committee (IAEC NO: (anti-diabetic drug) and is classified as a sulfonylurea. Its classification P16/VCP/IAEC/2012/3/VVR/AE2). has been ambiguous, as literature uses it as both a first-generation [10], and second-generation [11] sulfonylurea. Gliclazide was shown to Induction of experimental diabetes protect human pancreatic beta-cells from hyperglycemia- induced apoptosis [12]. It was also shown to have an antiatherogenic Male Wistar rats (200-250 gms) were fasted for 16 h before the effect (preventing accumulation of fat in arteries) in type 2 diabetes [13]. induction of diabetes with Streptozotocin (STZ), Animals (n=6) were Itopride is a prokinetic benzamide derivative unlike metoclopramide injected intra peritoneal with 0.22-0.25 ml of freshly prepared or domperidone. These drugs inhibit dopamine and have a gastro solution of STZ (60 mg/ml in 0.01 m citrate buffer, pH 4.5) at a final kinetic effect [14]. Itopride is indicated for the treatment of dose of 60 mg/kg body wt. The diabetic state was assessed in STZ- functional dyspepsia and other gastrointestinal conditions [15]. After treated rats by measuring the non-fasting serum glucose oral administration Itopride undergoes rapid and extensive absorption concentration after 48 h. Only rats with serum glucose levels greater with levels of itopride peaking in the blood plasma after only 35 min. than 300 mg/dl were selected and used in this experiment. Itopride is primarily eliminated via the kidneys having an elimination Study design [18] half-life of approximately 6 h [16]. The rats were grouped as follows To treat the coexisting disease, multidrug therapy is inevitable and there is every possibility for a drug interaction to occur when drugs Group I: Gliclazide (30 mg/kg, oral) alone in single dose/day in are concomitantly used [17]. diabetic rats up to 8 d.

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Group II: Itopride (20 mg/kg, oral) alone in single dose/day in retention time of an internal standard (phenformin), gliclazide and diabetic rats up to 8 d. itopride in plasma were 9.8, 7.2 and 3.3 min, respectively [21]. Group III: Itopride (20 mg/kg, oral) alone in single dose/day in Standard calibration curve of Gliclazideand Itopride in rat plasma normal healthy rats up to 8 d Different concentrations (0.05, 0.1, 0.5, 1, 5, 10, 20, 40 µg/ml) of Group IV: Gliclazide (30 mg/kg, oral) and Itopride concomitant gliclazide, itopride in plasma were prepared for calibration curve. administration in diabetic rats as Single dose/day up to 8 d. The samples were treated as above for protein precipitation method and peak areas of gliclazide and Itopride were noted down. The peak Collection of blood samples area ratios obtained at different concentrations of the gliclazide and Blood samples were collected after administration of Gliclazide 30 Itopride were plotted using UV–Vis detector at 238 nm. mg/kg, Itopride 20 mg/kg as per animal body weight on day 1 and Pharmacokinetic analysis day 8 blood samples of 1 ml were drawn from each anesthetized (isoflurane) rat at pre-determined time intervals was collected from Plasma concentration versus time data was analyzed using standard the retro-orbital plexus using a capillary tube into pre-labeled non-compartment analysis. AUC 0-t refers to the AUC from 0 to 24 h, eppendorf tubes containing 10% of K2EDTA anticoagulant ). which was determined by linear trapezoidal rule and AUC0-α refers to The time intervals for the sample collection were 0 (Pre dose), 0.5, 1, the AUC from time at zero hours to infinity. The AUC0-α was calculated (20 μl 2, 4, 6, 8 and 24 h (post dose), Equal amount of saline by oral route using the formula AUC0-t+[C last/K] where C last is the concentration in was administered to replace blood volume at every blood µg/ml at the last time point and K is the elimination rate constant. withdrawal time. Various pharmacokinetic parameters like area under the curve [AUC], Plasma was obtained by centrifuging blood samples by using elimination half life [t½]. Apparent volume of distribution (V/f) total refrigerated (REMI ULTRA) at 3000 rpm for 5 min. The obtained clearance (Cl/f) and mean residence time for each subject using a non- plasma samples were transferred into pre-labeled micro centrifuge compartmental analysis by using Win Nonlin 5.1 software. tubes and stored at 0 °C until analysis of pharmacokinetic Statistical analysis parameters. As described above, all the procedures were followed on day 8 also. Pharmacokinetic−2 parameters were calculated by non- Statistical comparisons for the pharmacokinetic study among, compartmental analysis by using Win Nonlin5.1 software [19, 20]. gliclazide, itopride, itopride alone and in combination groups and plasma concentration–response study among concentrations and time Method of analysis were carried out with student’s paired T-Test a value of P<0.05 was Preparation of plasma samples for HPLC analysis considered to be statistically significant. Data were reported as mean±SEM linear regressions were used to determine the relationship Rat plasma (0.5 ml) samples were prepared for chromatography by between total plasma concentrations and pharmacokinetic precipitating proteins with 2.5 ml of ice-cold absolute ethanol for parameters. The mean concentration versus time profile of gliclazide each 0.5 ml of plasma. After centrifugation the ethanol was and itopride in rat plasma is shown in fig. 1, 2, 3 and 4. transferred into a clean tube. The precipitate was suspended with 1 ml of acetonitrile by vortexing for 1 min. After centrifugation (5000– RESULTS AND DISCUSSION 6000 rpm for 10 min), acetonitrile was added to the ethanol and the organic mixture was taken to near dryness by a steam of nitrogen at In the present study, Gliclazide is completely absorbed after oral room temperature. Samples were reconstituted in 200 µ1 of mobile administration with peak plasma concentration of 11.54±2.14µg/ml phase were injected for HPLC analysis. after 2 h of dosing on day 1. In combination with Gliclazide and Itopride on day 1, the peak plasma concentration of Gliclazide For HPLC analysis size, mobile Phase 18.33±0.12µg/ml occurred at 2hr after dosing. There was no consisting of 0.05M Potassium Dihydrogen phosphate and 0.5% Tri significant increase in peak plasma concentration levels. Similarly Ethyl Amine (TEA) C18buffer column having with pH 2.7,5μm and particle Meth anol in the ratio of Itopride is completely absorbed after oral administration with peak 60:40 v/v. The flow rate was 0.8 ml/min and the effluents were plasma concentration 3.92±0.03µg/ml occurred at 2hr after dosing monitored at 238 nm. Internal standard Phenformin was used. The on day 1 in combination with Gliclazide and Itopride on day 1.

Table 1: Pharmacokinetic parameters of Gliclazide alone and in Combination with Itopride on day 1 parameters Gliclazide alone Gliclazide combination with Itopride Level of significance(p<0.05) Cmax (µg/ml) 11.54±2.14 18.33±0.12 NS tmax (h) 2±0 2±0 NS AUC o-t (µg/ml/h) 137.22±0.74 176.37±0.52 NS AUC o-inf (µg/ml/h) 158.82±2.50 158.15±1.85 NS t½ (h) 11.56±0.28 10.64±0.09 NS CL/f (ml/h/kg) 1.18±0.015 2.23±0.01 NS V/F (ml/kg) 25.28±0.26 37.56±0.25 NS NS: Not Significant

Table 2: Pharmacokinetic parameters of Gliclazide alone and in Combination with Itopride on day 8 Parameters Gliclazide alone Gliclazide combination with Itopride Level of significance (p<0.05) Cmax (µg/ml) 25.83±1.11 25.41±1.21 NS tmax (h) 2±1.26 2±1.14 NS AUC o-t (µg/ml/h) 156.371.24± 158.8±1.21 NS AUC o-inf (µg/ml/h) 178.15±1.45 196.12±1.31 NS t½ (h) 8.78±1.54 9.08±1.41 NS CL/f (ml/h/kg) 1.961±0.01 1.92±1.51 NS V/F (ml/kg) 26.67±0.52 27.89±1.25 NS NS: Not Significant

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Table 3: Pharmacokinetic parameters of Itopride in diabetic versus healthy male Wistar rats on day 1 Parameters Itopride in diabetic rats Itopride in healthy rats Level of significance (p<0.05) Cmax (µg/ml) 3.92±0.07 2.72±0.03 NS tmax(h) 2±0 2±0 NS AUC o-t (µg/ml/h) 42.49±11.1 21.9±0.118 NS AUC o-inf (µg/ml/h) 59.53±41.7 27.49±0.808 NS t½ (h) 12.22±15.1 12.03±1.11 NS CL/f(ml/h/kg) 15.475±12.2 26.71±0.18 NS V/F(ml/kg) 459.16±2.1 497.65±3.09 NS NS: Not Significant

Table 4: Pharmacokinetic parameters of Itopride in diabetic versus healthy male Wistar rats on day 8 Parameters Itopride in diabetic rats Itopride in non diabetic rats Level of significance (p<0.05) Cmax (µg/ml) 4.80±0.04 3.65±0.06 NS tmax(h) 2±0 2±0 NS AUC o-t (µg/ml/h) 44.11±0.225 38.22±0.09 NS AUC o-inf (µg/ml/h) 61.57±0.43 67.37±0.336 NS t½ (h) 12.49±0.186 18.92±0.009 NS CL/f(ml/h/kg) 13.01±0.08 11.88±0.042 NS V/F(ml/kg) 255.8±2.192 345.6±0.639 NS NS: Not Significant

Table 5: Pharmacokinetic parameters of Itopride alone and in Combination with Gliclazide in diabetic rats on day 1 Parameters Itopride alone Itopride with Gliclazide Level of significance(p<0.05) Cmax (µg/ml) 3.92±0.03 4.06±0.03 NS tmax(h) 2±0 2±0 NS AUC o-t (µg/ml/h) 33.5±0.20 41.63±0.20 NS AUC o-inf (µg/ml/h) 52.6±0.52 52.16±0.525 NS t½ (h) 15.23±0.27 12.57±0.27 NS CL/f(ml/h/kg) 15.43±0.26 19.03±0.26 NS V/F(ml/kg) 358.65±4.31 337.66±4.31 NS NS: Not Significant

Table 6: Pharmacokinetic parameters of Itopride alone and in Combination with Gliclazide in Diabetic rats on day 8 parameters Itopride alone Itopride with Gliclazide Level of significance(p<0.05) Cmax (µg/ml) 4.80±0.04 5.07±0.04 NS tmax(h) 2±0 2±0 NS AUC o-t (µg/ml/h) 44±0.225 51.6±0.25 NS AUC o-inf (µg/ml/h) 61.56±0.43 69.76±0.88 NS t½ (h) 12.49±0.18 12.89±0.29 NS CL/f(ml/h/kg) 13.01±0.08 13.48±0.03 NS V/F(ml/kg) 335.89±2.16 371.1±2.26 NS NS: Not Significant

Fig. 1: Mean Plasma concentrations (µg/ml) of Gliclazide alone Fig. 2: Mean Plasma concentrations (µg/ml) of Gliclazide alone and combination with Itopride on day 1 in diabetic male Wistar and combination with Itopride on day 8 in Diabetic male Wistar rats (n=6) rats (n=6)

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Volume of distribution was increased 3.8% and 1.72% in Gliclazide alone compared with Gliclazide and Itopride on day 1 and day 8 respectively. similarly Itopride in diabetic rats versus in healthy rats resulted 359.16 and 497.65 on day 1 respectively Itopride alone on day 1 and combination with Gliclazide on day 1 administration resulted in 1.5% increases of volume distribution (m1/kg) in alone Itopride group treated rats similarly on day 8 administration resulted in 5.5 increases of volume of distribution in alone Itopride treated rats. From the above results there is no significant difference of Cmax,tmax, AUC (0–t) and AUC (0-inf) , t½, Cl/f and V/f on day 1 and day 8 of Gliclazide and Itopride in both diabetic and healthy rats. Potentially the combination use of Gliclazide and Itopride is safe.

CONCLUSION Fig. 3: Mean plasma concentrations (µg/ml) of Itopride alone Considering the prevalence of diabetic gastroparesis as well as the and combination with Gliclazide on day 1 in diabetic male prescribing pattern of drugs in patients suffering from such Wister rats (n=6) complications, it is apparent that antidiabetic and gastroparesis drugs seem to be potential candidates for concomitant

administration. Gliclazide and Itopride are prescribed frequently together to the diabetic-gastroparesis patients. The present work was aimed to confirm the safety of concomitant administration of Gliclazide and Itopride in rats without any drug-drug interactions. There was no significant change in the serum levels and pharmacokinetics parameters of Cmax, tmax, AUC (0–t) and AUC (0-inf), t½, Cl/f and V/f on day 1 and day 8 of Gliclazide and Itopride in both diabetic and healthy rats. It may be concluded that during concomitant administration of Gliclazide and Itopride at therapeutic doses, drug–drug interaction does not occur. Therefore, the therapeutic dose and the frequency of administration of Gliclazide need not be adjusted in Gastroparesis patients with Diabetes.

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